INTRODUCTION:

Coronary heart disease (CHD) is one of the most investigated diseases in medicinal history. Cholesterol is an important component of the cell membrane and is essential for the synthesis of various important metabolites. HMGCoA Reductase (HMGCR), a key enzyme in the cholesterol biosynthesis, catalyzes the conversion of 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) into mevalonate.

Human HMGCR consists of polypeptide chains of 888 amino acids with three functional portions: Residues 1-339 span the membrane of the endoplasmic reticulum eight times; residues 340-459 connect the membrane portion to the catalytic portion (residues 460-888), which resides in the cytoplasm. This enzyme is anchored in the membrane of the endoplasmic reticulum, having seven transmembrane domains, with the active site located in a long carboxyl terminal domain in the cytosol¹. The inhibition of this enzyme results in the significant decrease in cholesterol levels²and thus reduces the risks of stroke and mortality³.

Normally in mammalian cells this enzyme is suppressed by cholesterol derived from the internalization and degradation of low density lipoprotein (LDL) via the LDL receptor as well as oxidized species of cholesterol. Thus this enzyme is the target of the widely available cholesterol-lowering drugs known collectively as the statins⁴. Bioinformatics involves the organization of data generated from experiments into databases, the development of new algorithms and software, using it for the interpretation and analysis of data. In the present study, isolated compounds from Cassia fistula bark were screened with the database of available structures, and algorithms adapted from artificial intelligence applications to understand the applicability of the docking tool. The compounds thus screened with docking tools provide a preliminary data for refinement of the chemical structures or modification of the specific site on a target protein or nucleotide, enabling binding at the site that is being modelled computationally with several different techniques. The availability of the structural information aids in proper selection of target for inhibitor discovery as the binding sites of the molecules are identified⁵.

MATERIALS AND METHODS:

Plant material:

The whole plant of Cassia fistula was collected from Seshachalam hills, Tirupati, Chitoor district, Andhra Pradesh in the month of January and was identified and authenticated by Dr. K. Madhava chetty. The powdered plant material was successively extracted in 500 ml of ethyl acetate and methanol using simple distillation⁶. The extracts were finally stored in air tight containers for further use.

In vitro HMG CoA reductase inhibitor activity:

HMG-CoA reductase inhibitory activity of the plants was determined based on spectrophotometric measurements. The HMG-CoA reductase assay kit was purchased from Sigma-Aldrich Co. (St Louis, MO, USA). The concentration of the HMG-CoA reductase stock solution was 0.5–0.75 mg/mL. Each crude extract (50 μg) was mixed with a reaction mixture containing nicotinamide adenine dinucleotide phosphate (400 μM), HMG-CoA substrate (400 μM), and potassium phosphate buffer (100 mM, pH 7.4) containing potassium chloride (120 mM), ethylenediaminetetraacetic acid (1 mM), and dithiothreitol (5 mM), followed by the addition of HMG-CoA reductase (2 μL). The reaction was incubated at 37°C, and absorbance was measured at 340 nm after 10 minutes^7,8. Simvastatin (Sigma-Aldrich Co.) was used as a positive control, and distilled water as a negative control. The HMG-Co A reductase inhibition (%) was calculated using the following formula:

Abs of control-Abs of treated group

% Inhibition =^{-------------------------------------------------------------------------------------------------------------------------}×100

Abs of control

Docking:

The docking analysis of the compounds with HMGCoA reductase (HMGCR) was carried out by Ligand Fit of Maestro 9.1 (Schrodinger Software Inc.). The software allows us to virtually screen a database of compounds and predict the strongest binders based on various scoring functions. The collection of enzyme substrate complexes were identified via docking and their relative stabilities were evaluated using their binding affinities. LigandFit was used for accurately docking ligands into protein active sites employing a cavity detection algorithm. A shape comparison filter is combined with a Monte Carlo conformational search for generating ligand poses consistent with the active site shape. Candidate poses are minimized in the context of the active site using a grid based method for evaluating protein-ligand interaction energies. The method appears quite promising, reproducing the X-ray structure ligand pose within an RMSD of 2A. A high-throughput screening study applied to the HMGCoA reductase receptor is also presented in which LigandFit, when combined with LigScore, an internally developed scoring function, yields very good hit rates for a ligand pool seeded with known actives⁹.

Docking Protocol:

Protein Preparation:

The crystal structure of HMG-CoA 1HWK was prepared and the active site was identified. The ligands and crystallographic water molecules were removed from the protein and the chemistry of the protein was corrected for missing hydrogen. Crystallographic disorders and unfilled valence atoms were corrected using alternate conformations and valence monitor options. Following the above steps of preparation, the protein was subjected to energy minimization using the CHARMm force field.

Ligand Preparation:

The methanolic extract of the Cassia fistula was analyzed using GCMS technique and many compounds were identified out of which three compounds were identified. These secondary metabolites were used as the ligands. The three dimensional structures of compounds were downloaded in .sdf format from PubChem and Chemdraw database. Hydrogen Bonds were added and the energy was minimized using CHARMm force field. G score, Hydrogen bond, Rotatable bond penalty, lipophilic term derived from hydrophobic grid for the active ligands were noted (Table 2).

Docking Studies:

The active site of the protein was first identified and it is defined as the binding site. The binding sites were defined based on the ligands already present in the PDB file (i.e. statin binding site region) which were followed by site sphere definition. The determination of the ligand binding affinity was calculated using LigScore and PLP1, JAIN and Dock score were used to estimate the ligand-binding energies. Apart from these, other input parameters for docking were set as default options.

RESULTS:

In vitro HMG CoA Reductase inhibitor activity:

Extracts of Cassia fistula bark was evaluated for HMG CoA Reductase inhibitor activity and was found that methanolic extract of Cassia fistula bark inhibited HMG-CoA reductase by 43% inhibition at 100 µg/mL, and 85% inhibition at 500 µg/mL. Ethyl actetate extract of Cassia fistula bark inhibited HMG-CoA reductase by 20% inhibition at 100µg/mL and 32% inhibition at 500 µg/mL. This shows that methanolic extract was found to be more potent when compared to that of ethyl acetate extract. Standard Pravastatin at 100 µg/mL, showed 65 % inhibition.

Table 1: Percentage inhibition of HMG CoA Reductase

Concentration	% inhibition	IC₅₀µg/ml
Blank	0
Methanol Extract (100 µg/ml)	43	150 µg/ml
Methanol Extract (500 µg/ml)	85	150 µg/ml
Ethyl acetate extract (100 µg/ml)	20	250 µg/ml
Ethyl acetate extract (500 µg/ml)	32	250 µg/ml
Standard (Pravastatin)	65

Docking results:

Table 2: Schrodinger XP Docking Scores

Title 1HWK	Glide g score	XP H Bond	XP Rot Penal	XP Lipophilic EvdW
Compound 2	-13.0197	-7.49186	0.06352	-1.71644
Atorvastatin	-12.5368	-3.74808	0.197351	-4.02509
Compound 3	-11.6776	-5.99898	0.078627	-2.64474
Compound 1	-10.3014	-5.4129	0.112962	-1.07652

G score= glide score, Lipophilic EvdW= Lipophilic term derived from hydrophobic grid potential,H bond= hydrogen bonding term, Rot Penal= Rotatable bond penalty.

Figure 1: Hydrogen bonding interactions of Compound 1 with PDB ID: 1HWK docking simulations in Glide 5.6

Compound 1 (total score -10.3014) demonstrated hydrogen bonding interactions with Glu 559, Asn 755, Asp 767, Asp 690, Arg 590, Asnn 658, Lys 691.

Figure 2: Hydrogen bonding interactions of compound 2 with PDB ID: 1HWK

Compound 2 (total score -13.0197) demonstrated hydrogen bonding interactions with Glu 559, Asn 755, Lys 691, Asp 690, Arg 590, Glu 665, Ser 661, Asn 658.

Figure 3: Hydrogen bonding interactions of Compound 3 with PDB ID: 1HWK

Compound 3 (total score -11.8889) demonstrated hydrogen bonding interactions with Gly 560, Lys 735, Ala 525, Asn 658, Argg 590, Asp 690.

Figure 4: Hydrogen bonding interactions of Atorvastatin with PDB ID: 1HWK

Atorvastatin (total score -12.5368) demonstrated hydrogen bonding interactions with Glu 559, Ser 565, Asn 755, Lys 691, Asp 690, Arg 590, Lys 692, Ser 684.

In vitro HMG CoA reductase inhibition:

It is well accepted that enzymes are the major regulators of the lipid metabolism; wherein HMG-CoA reductase is one of the most clinically important enzymes involved in the cholesterol biosynthetic pathway. HMG CoA reductase is the rate limiting enzyme in the cholesterol biosynthetic pathway. It converts HMG CoA to mevalonate, so HMG CoA reductase inhibition would prevent the formation of mevalonate which in turn would decrease the sterol biosynthesis. Changes in the reductase activity are closely related to changes in the overall rate of cholesterol synthesis. This suggests that the inhibition of HMG-CoA reductase would be an effective mean to lower plasma cholesterol¹⁰. Thus, this enzyme is the target of the widely available cholesterol lowering drugs known, collectively, as statins⁴. Although, most of the HMG-CoA reductase inhibitors have some adverse side effects^11,12. In vitro HMG CoA reductase inhibition study has shown that the methanolic and ethyl acetate extract of Cassia fistula could inhibit the HMG CoA reductase. This elucidates the mechanism of action by which the Cassia fistula possesses antihyperlipidemic activity.

Docking results:

Results demonstrated that the compound 2 has shown maximum interactions when compared to that of other compounds. It was also seen that the compound 2 has similar interactions with the same amino acids as that of the standard. It has got the highest g score (-13.0197) than that of atorvastatin (-12.5368) which showed that the mechanism of action of the Cassia fistula by which it shows anti hyperlipidemic activity is by inhibiting the HMG CoA enzyme.

Furthermore, to understand the binding mechanism and corroborate our in vitro hypothesis, in silico molecular interaction studies were conducted between HMG-CoA reductase and the inhibitor compounds. The docked structure of compounds-HMG-CoA reductase involved contributions Glu 559, Asn 755, Asp 767, Asp 690, Arg 590, Asn 658, Lys 691.residues, which were not from the catalytic domain of the enzyme. This confirms the uncompetitive mode of inhibition observed in vitro. The result of interaction study of standard drug atorvastatin with HMG-CoA reductase is in consensus with our previously published data. Our results are in concordance with earlier reports demonstrating that the reduction in HMG-CoA reductase activity is responsible for the hypolipidemic property of natural agents¹³. This lipid lowering activity might be due to the pleiotropic effect that the compound exhibits through reduced HMG-CoA reductase activity¹⁴. The compound 2 showed better activity than drug atorvastatin, marked hypolipidemic property and might be a drug candidate.

CONCLUSION:

Understanding the interactions between proteins and ligands is crucial for the pharmaceutical and functional food industries. The emergence of bioinformatics has offered a platform to explore diseases at molecular level using computational tools. The Protein-Ligand interaction plays a significant role in structure based drug designing. The experimental structures of these protein/ligand complexes are usually obtained, by time-consuming techniques such as X-ray crystallography or NMR. These screening methods are routinely and extensively used to reduce cost and time of drug discovery. In the present work, the enzyme HMG CoA Reductase and the ligand molecules were taken to explore the binding mechanism of identified plant compounds from GC-MS study to the HMG CoA Reductase enzyme. Therefore, this study emphasizes the importance of small molecules from Cassia fistula their use to enhance protein-ligand interaction studies in silico. Experimental works has already proved to possess Antihyperlipidemic activity which is further correlated with docking studies. Further work can be extended to study the receptor-ligand interactions experimentally based on screening, docking and consensus scoring techniques.

ACKNOWLEDGEMENTS:

The authors would like to acknowledge their gratification to the Principal and Management for their assistance and guidance with this paper.

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Received on 13.11.2018 Modified on 01.12.2018

Asian J. Research Chem. 2019; 12(2): 89-93.

DOI: 10.5958/0974-4150.2019.00020.8